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Gateway庐 Cloning Technology
TABLE OF CONTENT
PRODUCT DESCRIPTION
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
Gateway Donor Vectors
Gateway Entry Vectors
Gateway Destination Vectors
Gateway Vector Conversion cassettes
Sequences of the Att Sites
The BP Recombination Reaction
PCR product recombination into the DONOR vector
Amount of DNA to be used in a BP reaction
The BP Clonase and reaction conditions
Information on pEXP7-Tet
The LR Recombination Reaction
The LR Clonase and reaction conditions
Information on pENTR-gus
Primers for sequencing Entry Clones
Sequencing the shRNA from pENTR/U6 or pENTR/H1/TO
Primers for sequencing Expression Clones
ALTERNATE PRODUCTS & COMPATIBILITY
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBER
COMPONENTS
Enzymes needed
Competent E. coli
Donor vectors
Entry vectors
Destination vectors
ASSOCIATED PRODUCTS
1
PRODUCT DESCRIPTION
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How Gateway Technology Works
Gateway Technology uses lambda phage-based site-specific recombination instead of restriction endonuclease and ligase
to insert a gene of interest into an expression vector. The DNA recombination sequences (attL, attR, attB, and attP) and
the Clonase enzyme mixtures (i.e. LR or BP Clonase) mediate the lambda recombination reactions.
General Gateway Cloning Recombination Notes
The Gateway Cloning Technology takes advantage of the well-characterized bacteriophage lambda-based site-specific
recombination instead of restriction enzymes and ligase. The power of the Gateway Cloning Technology is that genes
cloned into Entry vectors can be subcloned in parallel into one or more Destination Vectors in a simple, 60-minute
reaction. Moreover, a high percentage (> 95%) of the colonies obtained carry the Expression Clone in the desired
orientation and reading frame.
Illegitimate recombination does not occur since Gateway Cloning does not operate by homologous recombination and
recombination with genomic sequence is predicted to be a rare event.
The nomenclature for the att sites used by Invitrogen is consistent with the lambda nomenclature. The only deviations are
in the attB1 or attB2 sites since these are mutant versions of the attB site that do not exist in lambda.
The recombination sites used in Gateway Cloning are not wild-type sites. Several point mutations were engineered into
the wild type att sites to generate novel specificities. For example attB1 only recombines with attP1 and not with wild
type attP or attP2. As these sites differ by only a few nucleotides, the specificities of the att sites for the paring partners is
extremely high.
There are certain limitations with regards to Gateway Cloning, both imposed by biology. The gene-of-interest will
always be connected to att sites, either attL (100 bp) in an Entry clone, or attB (25 bp) in Expression clones. Therefore it
needs to be decided initially whether elements such as eukaryotic or prokaryotic translation signals, or a 3' stop codon
need to be included before proceeding with the generation of the Entry clone. It is best to construct two Entry Clones;
one with the stop codon after the coding sequence for N-terminal fusions, and one without the stop codon for C-terminal
fusions.
The exact minimum size limit that can be used in a Gateway reaction is not known. There will be a minimal size limit,
probably constrained by the topology of the recombination reaction. Early data suggests that 100 bps between the att
sites may be sufficient.
Recombination Enzymes involved in the Gateway recombination reactions
Lambda recombination is catalyzed by a mixture of enzymes that bind to specific sequences (att sites), bring together the target
sites, cleave them, and covalently attach the DNA. The Clonase enzyme mixtures utilize a combination of the bacteriophage 位
Integrase (Int) and Excisionase (Xis) proteins and E. coli Integration Host Factor (IHF) proteins.
Int:
Has type I topoisomerase activity. Cuts and reseals the att sites via covalent Int-DNA intermediate
Binds specifically to 2 different families of DNA sequences:
1. core: CAACTTNNT
2. arm: C/AAGTCACTAT
Required for both excision (LR reaction) and integration (BP reaction) of phage
Xis
Required for excision (LR) but not integration (BP) of phage.
Inhibits integration (BP) at physiological conditions
Relatively stable in vitro but rapidly degraded in cells
Promotes efficient LR recombination in presence of Int and IHF
No enzymatic function but rather sequence-specific cooperative binding to adjacent sites in the P arm thus introducing
sharp bend in the DNA.
Also associated with cooperative interactions with DNA-bound Int .
IHF
E. coli-derived protein as opposed to phage-derived (Xis & Int)
Essential for both excision and integration (LR and BP reactions respectively)
Heterodimer composed of alpha and beta subunits
Similar to other type II DNA binding proteins such as histones
2
No known enzymatic function but it binds to and bends DNA at specific sites
SHIPPING CONDITIONS
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Gateway Donor, Entry, and Destination vectors are shipped in a supercoiled and lyophilized format with a few
exceptions. The pENTR-Gus positive control is supplied supercoiled in TE buffer whereas pEXP7-tet is supplied
linearized in TE buffer. The Yeast two-hybrid vectors, pDEST22 and pDEST32 are supplied as linearized vectors in TE
buffer. The adenoviral destination vectors are shipped supercoiled in TE buffer.
All lyophilized vectors are shipped at room temperature and all the vectors in solution are shipped on dry ice. The
competent E. coli, and enzymes are shipped on dry ice.
STORAGE CONDITIONS
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Store all Gateway vectors at 鈥?20oC.
BP and LR enzyme mix must be stored at 鈥?80o C.
5X BP or LR Reaction buffer and the Proteinase K solution can be stored at 鈥?80oC or 鈥?20oC.
BP Clonase II and LR Clonase II enzyme mixes can be stored at 鈥?20oC or 鈥?80oC.
STABILITY
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All reagents are guaranteed stable for 6 months when properly stored.
Both BP, and LR Clonase enzyme mixes are stable for at least 6 month from date of purchase.
When used in a reaction, the mix is first thawed on ice for two minutes, mixed gently by tapping or vortexed very briefly.
After taking the desired aliquot out, the mix should be returned to 鈥?80oC promptly.
The enzyme mix retains 50% activity after 15 cycles of freeze-thaw. It is also stable (100 % activity) overnight at 4oC or
one week at 鈥?20oC. It is not recommended to aliquot the mix since this can lead to loss of activity.
Proteinase K is stable for 12 months at 4掳C as a powder and up to 2 weeks at room temperature as a powder or solution.
QC SPECIFICATIONS
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Gateway Vector Conversion cassettes: The cassettes functionally tested with the ccdB assay as part of the QC procedure.
Gateway BP Clonase Enzyme Mix: Functionally tested in a 1 hour recombination reaction followed by gel electrophoresis
analysis and a transformation assay.
All pDONR vectors:
A BP cloning reaction, and the ccdB assay is done as part of the QC procedure.
pENTR D-TOPO Vector:
TOPO Cloning with pENTR/D-TOPO and a directional test PCR product must yield the following results when tested using the
control conditions listed in the manual:
(1) pENTR/D-TOPO and directional PCR product ligation: cloning efficiency must be > 85% as based on colony counts
from plus insert plates and vector only plates.
(2) Directional PCR to confirm directional cloning of product: > 36 out of 40 transformants analyzed from plus insert
plates must contain the test PCR product cloned in the correct orientation.
PROTOCOL AND APPLICATION NOTES
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Gateway Donor Vectors
3
Gateway Entry Vectors
Gateway Destination Vectors
Gateway Vector Conversion cassettes
Sequences of the Att Sites
The BP Recombination Reaction
PCR product recombination into the DONOR vector
Amount of DNA to be used in a BP reaction
The BP Clonase and reaction conditions
Information on pEXP7-Tet
The LR Recombination Reaction
The LR Clonase and reaction conditions
Information on pENTR-gus
Primers for sequencing Entry Clones
Sequencing the shRNA from pENTR/U6 or pENTR/H1/TO
Primers for sequencing Expression Clones
Gateway Donor Vectors
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Inserts can be released from all pDONR vectors with BsrG1, which cuts in both attL sites and whose recognition
sequence is TGTACA. An exception is pDONR P4/P1R that is part of the MultiSite Gateway System and has an attL4
site whose sequence is different from attL1 and attL2.
Donor vectors contain two transcription termination sequences (rrnB T1 and T2) upstream from attP1. This prevents
transcription of genes cloned into pDONR vectors from other vector-encoded promoters thereby reducing possible toxic
effects.
The minimum insert successfully cloned into a pDONR vector in-house was 70bp and largest was 12 Kb. Although,
successful cloning of small inserts from 50-200 bp is sequence dependent.
Although pDONR201 and pDONR207 contain a pUC ori, they replicate less efficiently resulting in lower plasmid yields.
In contrast pDONR221 acts as a high-copy number plasmid typically yielding 0.5 - 1.0 mg of DNA per liter.
Gateway Entry Vectors
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Inserts can be released from all pENTR vectors with BsrG1, which cuts in both the attL sites and whose recognition
sequence is TGTACA.
The Entry vectors 1A, 2B, 3C, 4, 11 contain the same vector backbone (outside the attL sites) and differ only in the
sequences and cloning sites provided between the attL sites. They contain a modified pUC origin of replication and
are low- to mid-copy number plasmids. In terms of DNA yield, these vectors are more like those with pBR322 ori.
All Entry vectors contain two transcription termination sequences (rrnB T1 and T2) upstream from attL1. This
prevents read-through transcription from other vector-encoded promoters, thereby reducing possible toxic effects.
The Shine-Dalgarno sequence in pENTR/SD/D-TOPO does not adversely affect mammalian expression when used
in an appropriate mammalian DEST vector. Hence this vector may be substituted for pENTR/D-TOPO.
When cloning into any of the Directional TOPO Entry vectors it is recommended to use molar ratios of 0.5-2:1 of
insert: vector. Too much PCR product will inhibit the cloning reaction; hence the PCR product may need to be
diluted 10 fold before cloning. Use 1-5ng of a 1 Kb product or 5-10ng of a 2 Kb product.
When cloning into pENTR/U6 or pENTR/H1/TO, the two synthesized DNA oligos do not need phosphate groups at the
5鈥? end since both these vectors have the 5鈥? phosphate groups.
The pCR8/GW/TOPO entry vector uses spectinomycin for selection; hence the entry clone generated can be used
with any destination vector. Most destination vectors are ampicillin-resistant, but there may be some that are
kanamycin or zeocin-resistant.
pCR8/GW/TOPO vector can be used to TA clone a PCR product amplified with any Taq DNA polymerase or
Invitrogen鈥檚 Platinum Taq DNA polymerase High Fidelity (catalog #11304-011).
Gateway Destination Vectors
4
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Inserts can be released from all DEST vectors with BsrG1, which cuts in both attB sites and whose recognition
sequence is TGTACA.
All the destination vectors have a pUC ori (for prokaryotic), SV40 ori (for mammalian), and 2碌 ori (for yeast).
When propagating pDONR, pENTR, and pDEST vectors with the ccdB gene, the E. coli can be grown at 30oC to
prevent random deletions of the gene. If this happens, the amount of background colonies will increase since the
selection method has been eliminated. It is recommended to verify the functionality of the ccdB gene by propagating
the Gateway vectors using selection on 20-30 ug/ml chloramphenicol plates.
Restriction enzymes used to linearize Destination vectors
Linearized Destination Vector can be obtained by cleaving at a restriction site within the region of the GATEWAY Cassette,
taking care to avoid the ccdB gene. All Destination Vectors from Invitrogen used to be provided linearized in this manner.
Although Invitrogen previously recommend using a linearized destination vector for more efficient recombination, further testing
at Invitrogen has found that linearization is NOT required to obtain optimal results for downstream application.
Vector Restriction Enzyme Used to Linearize It
pDEST 14 Mlu I
pDEST 15 BssH II
pDEST 17 BssH II
pDEST 8 Mlu I
pDEST 10 Mlu I
pDEST 20 EcoR I
pDEST 12.2 Mlu I
pDEST 22 BssH II
pDEST 26 EcoR I
pDEST 27 EcoR I
pDEST 32 BssH II
pET-DEST42 NcoI
pT-Rex-DEST30 EcoRI
pT-Rex-DEST31 EcoRI
pcDNA-DEST40 EcoRI
pcDNA-DEST47 EcoRI
pMT-DEST48 EcoRI
pYES-DEST52 EcoRI
pBAD-DEST49 EcoRI
pEF-DEST51 EcoRI
pcDNA-DEST53 EcoRI
Gateway Vector Conversion cassettes
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All the Conversion cassettes are blunt-ended and 5鈥?-phosphorylated.
Each reading frame cassette has a different unique restriction site between the chloramphenicol resistance and ccdB genes
(Mlu I for the reading frame A cassette, Bgl II for the reading frame B cassette, and Xba I for the reading frame C
cassette).
The reading frame of the fusion protein domain must be in frame with the core region of the attR1 site for an N-terminal
fusion (e.g. the six As are translated into two lysine codons). For a C-terminal fusion protein, translation through the core
region of the attR2 site should be in frame with 鈥揟AC-AAA-, encoding -Tyr-Lys-. For native proteins, any of the three
Gateway Cassettes may be used since there will be no translation through the att sites. Therefore reading frame issues
through the att sites are not relevant.
5
For sequencing from within an attR1 region the GW3 priming site primer can be used. The primer sequence is 5鈥?-TTA
ATA TAT TGA TAT TTA TAT CAT TTT ACG-3鈥?. The primer anneals about 30 bp downstream of the 5鈥? end of the
attR1 site.
When sequencing, as long as the attR sites are intact, a restriction enzyme with a site very close to attR sites can be used
to linearize the Gateway-converted vector.
Sequences of the Att Sites
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Of the 4-att sites, attP is the most complex and attB is the simplest. The prophage sites attL and attR are hybrids of attP and attB.
Contribution of attL to attP is in blue.
Contribution of attR to attP is in green.
Contribution of attL and attR to attB is in yellow.
attL1: 100bp CAAATAATGA TTTTATTTTG A CTG ATA GTG ACC TGT TCG TTG CAA CAA ATT GAT
AAG CAA TGC TTT TTT ATA ATG CCA ACT TTG TAC AAA AAA GCA GGC T
AC CCA GCT TTC TTG TAC AAA GTT GGC ATT ATA AGA AAG CAT TGC TTA TCA ATT
attL2:
100bp TGT TGC AAC GAA CAG GTC ACT ATC AGT CAA AAT AAA ATC ATT ATT TG
ACA AGT TTG TAC AAA AAA GCT GAA CGA GAA ACG TAA AAT G ATA TAA ATA TCA
attR1:
125 bp ATA TAT TAA ATT AGA TTT TGCATAAAAA ACAGACTACA TAATACTGTA
AAACACAACA TATCCAGTCA CTATG
CAT AGT GAC TGG ATA TGT TGT GTT TTA CAG TAT TAT GTA GTC TGT TTT TTA TGC
attR2:
125 bp AAA ATC TAA TTT AAT ATA TTG ATA TTT ATA TCA TTT TAC GTT TCT CGT TCA GCT
TTC TTG TAC AAA GTG GT
CAAATAATGA TT TTA TTT TGA CTG ATA GTG ACC TGT TCG TTG CAA CAA ATT GAT
attP1:
233 bp GAG CAA TGC TTT TTT ATA ATG CCA ACT TTG TAC AAA AAA GCT GAA CGA GAA
ACG TAA AAT GAT ATA AAT ATC AAT ATA TTA AAT TAG ATT TTG CAT AAA AAA
CAG ACT ACA TAA TAC TGT AAA ACA CAA CAT ATC CAG TCA CTA TGA ATC AAC
TAC TTA GAT GGT ATT AGT GAC CTG TA
TA CAG GTC ACT AAT ACC ATC TAA GTA GTT GAT TCA TAG TGA CTG GAT ATG TTG
attP2:
233 bp TGT TTT ACA GTA TTA TGT AGT CTG TTT TTT ATG CAA AAT CTA ATT TAA TAT ATT
GAT ATT TAT ATC ATT TTA CGT TTC TCG TTC AGC TTT CTT GTA CAA AGT TGG CAT
TAT AAG AAA GCA TTG CTT ATC AAT TTG TTG CAA CGA ACA GGT CAC TAT CAG
TCA AAA TAA AATCAT TAT TTG
ACA AGT TTG TAC AAA AAA GCA GGC T
attB1
AC CCA GCT TTC TTG TAC AAA GTG GT
attB2
Note: Not all att sequences are conserved in every vector; att sites have been modified in various
vectors to increase efficiency, minimize secondary structure, etc. Thus, an attB1 site in one vector
may not be identical to an attB1 site in another vector (but we still call them both attB1 sites). Only
the 21-bp consensus sequence between all att sites is crucial for recombination.
For attB1 the essential sequence is: 5鈥?- CNNNTTTGTACAAAAAANNNG.
For attB2 the essential sequence is: 5鈥?- CNNNTTTCTTGTACAAANNNG.
Changes to other base pairs do not affect recombination.
The attB amino acid sequence does not interfere with transcription or translation. No effect of the attB sites on
expression levels in E. coli, insect and mammalian cells have been observed.
Simpson et al. EMBO Reports 11(31): 287-292, 2000 demonstrated that GFP fusions localized to the proper intracellular
compartment. The proteins contained the attB1 or attB2 sequences.
It is believed that there may be certain mutation-prone hotspots within the attL sites that happen in E.coli. However some
of these hotspots do not interfere with the recombination reaction nor do they cause a shift in the reading frame of the
GOI if recombined into a N-terminal tagged destination vector. It is not known why these hotspots occur; when it does, it
does not get transferred into the destination vector but remains within the backbone of the Entry clone.
An article that describes the specificity of att sites is Sasaki et al. J. Biotechnol. 2004; 107(3):233-43. Evidence for high
specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system.
6
Schematic of the creation of attB sites after LR recombination
Contribution of attR to attB is in green.
Contribution of attL to attB is in blue.
Integrase (Int) produces a seven-base staggered cut during the recombination reactions indicated by the arrows in the
figure.
There are 8 amino acids (letters above triplet codons) contributed by the attB site, which get added to the 5鈥? or 3鈥? end of
the gene of interest depending on the location of the fusion tag in the destination vector.
The BP Recombination Reaction
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Recombination reaction between an Expression Clone or PCR product (containing a 鈥済ene鈥? flanked by attB1 and attB2
sites) and pDONR (containing attP1 and attP2 sites) to generate an Entry clone that now contains the 鈥済ene鈥? of interest
flanked by attL1 and attL2 sites.
Our studies have shown that the BP recombination reaction is approximately 5-10 fold more efficient than a ligation
reaction to clone a piece of DNA. Approximately 5-10% of the starting material is converted into product during a BP
reaction.
The largest PCR fragment cloned in-house is 10 Kb (see Table below). In theory a much larger fragment can be cloned.
A Gateway Cloning reaction is essentially swapping one fragment out of a plasmid and replacing it with another where
the reaction cannot discriminate between the "vector" and the "insert". Gateway reactions have been performed in-house,
using a Destination Vector that was approximately 130 Kb, so in theory large inserts of that size can be transferred via
Gateway technology.
High-efficiency cloning of large genes using pDONR donor vector
PCR products (0.26 Kb to 10.1 Kb) were cloned into the pDONR donor vector. Random colonies were selected and screened
for the presence of insert and orientation. The range of PCR fragments demonstrated >90% cloning.
Size (Kb) PCR DNA (fmol) PCR DNA (ng) Colonies/ml Correct clones/Total
Transformation* clones examined
10/10#
0.26 15 3 1223
38 7.5 2815
1.0 15 10 507 49/50
38 25 1447
1.4 15 14 271 48/50
38 35 683
3.4 15 34 478 9/10#
38 85 976
4.6 15 46 190 10/10#
38 115 195
6.9 15 69 30 (235鈥?) 47/50
38 173 54 (463鈥?)
7
10.1 7.5 50.5 16 (112鈥?) 15/16
37.5 252.5 42 (201鈥?)
* pUC+ 108 CFU/ml
鈥? After overnight incubation
# DNA mini-prep analysis
PCR product recombination into the pDONR vector
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The best place to include a protease cleavage site or any other N-term tag is between the attB1 sequence and the first gene
specific codon (in most cases, ATG). An example of adding a TEV Protease site:
5'-ACA-AGT-TTG-TAC-AAA-AAA-GCA-GGC-TNN-GAA-AAC-CTG-TAT-TTT-CAG-GGC-ATG-forward gene
specific sequence-3'
The sequence as describe above would generate a protein with one additional amino acid (glycine) on the N-terminus
after cleavage with TEV. An alternative would be to replace the ATG with the GGC codon. This would generate a
protein with a glycine residue in place of the methionine residue after cleavage with TEV protease.
For purity of the attB-containing primers, 50 nmol of standard purity oligos are adequate for most applications. For
cloning smaller products, purifying the oligos by Cartridge or PAGE doesn't significantly increase colony output (not
more than 2-3 fold). However, for cloning large PCR products (> 5kb), colony output can be increased if the oligos
(when >65 bases) are further purified (i.e. Cartridge or PAGE). The oligos should be dissolved to a concentration of 20-
50 碌M.
The 4-G鈥檚 at the 5鈥? end of the attB primer sequences are necessary for the BP reaction and cannot be replaced by
analogous sequences. It is believed that this stretch of Gs serve as a substrate in the BP reaction by allowing more of an
area for protein binding; although this has not been directly demonstrated. Addition of more than 4Gs inhibits the BP
recombination reaction due to the formation of an inhibitory secondary structure. The next two bases after the G cannot
be AA, AG or GA since this would form a stop codon.
Typically the attB sequences on PCR primers are not a problem during PCR amplification. Hence there is no need to
change the PCR reaction conditions when primers have the attB sequence compared to reactions using gene specific
primers alone. It is recommended that the attB-PCR product be cleaned up with a PEG precipitation step. This removes
PCR buffer, unincorporated dNTPs, and primer dimers. Small primer-dimers clone very efficiently and decrease the
number of correct clones whereas leftover PCR buffer may inhibit the BP reaction.
One-Step Adapter PCR method for HTP Gateway cloning: For detailed protocol see Quest 1.2; pg 53-55. One can add
the att B adaptors by using the 4 primers all in one tube. The best ratio of the first gene specific and the second attB
primers is 1:10. The protocol is:
Template DNA 50ng
10x Pfxb amplification buffer 1ul
10x PCRx enhancer solution 1ul
Gene specific primers 2 pmol each
attB primers 20 pmol each
Platinum Pfx 1unit
Total volume 10ul
PCR product can be used in BP reaction without any purification, and around 90% clones were converted
Gateway entry clones.
Amount of DNA to be used in a BP reaction
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For the most efficient reaction, it is best to not have attB sites in molar excess of attP sites. For a 20 碌l reaction, 300 ng
(no more than 500 ng) of the donor vector is recommended. Using too much of the donor vector in the reaction tube will
inhibit the BP reaction and also result in intact donor vector being co-transformed with the Entry Clones. This will
reduce the amount of colonies on the plate by killing the transformed E. coli due the presence of the ccdB gene.
For PCR products > 4 Kb, the number of colonies obtained per fmol of PCR DNA added decreases with increasing size.
Thus for larger PCR products it is recommended to increase the amount of DNA to at least 100 fmol of PCR product per
8
20 碌l reaction, and using incubations longer than one hour (i.e. 6 hours or overnight). The largest PCR-amplified DNA
cloned in-house was 10.1 Kb.
The standard BP Reaction uses 300 ng of pDONR Vector and 30-300 ng attB-flanked PCR product or Expression Clone
for 1 hour at 25oC. Longer incubation times of up to 24 h can be used to convert a higher percentage of starting attB-DNA
to product. Longer incubations are recommended for PCR products 鈮?5 Kb; however in these cases the number of
colonies will be decreased . Increasing the incubation to 4-6 h will typically increase colony output 2-3 fold and 16-24 h
will typically increase colony output 5-10 fold (Focus 18.1: pg 27).
The BP Clonase and reaction conditions
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The BP clonase mix contains Int (Integrase), and IHF (Integration Host Factor).
BP Clonase II contains enzymes and buffer in a single mix to enable convenient ten-microliter reaction set up with fewer
pipetting steps whereas the original BP Clonase requires addition of enzyme and buffer.
If Proteinase K is not added after the BP reaction, there could be a 10-fold decrease in efficiency.
The one-tube protocol with the new BP, and LR Clonase II Enzyme mix is slightly different from the original one-tube
protocol since the enzymes and buffer are in same enzyme mix. Refer to the Gateway Technology with Clonase II
manual.
Information on pEXP7-Tet
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This is the positive control for the BP recombination reaction, which permits a tet-resistance cassette (1.4 kb) to be cloned
into the donor vector. It is an approximately 5.76 Kb linearized plasmid DNA that has a 1.4 kb segment containing attB
sites flanking the tet-resistance gene and its promoter.
The LR Recombination Reaction
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Recombination reaction between an Entry clone (containing a 鈥済ene鈥? flanked by attL1 and attL2 sites) and a DEST
vector (containing attR1 and attR2 sites) to generate an Expression clone that now contains the 鈥済ene鈥? of interest flanked
by attB1 and attB2 sites. The recombination requires the LR Clonase Enzyme.
Up to 30% of the starting material is converted to product during an LR reaction.
The possibility of generating PCR products with attL sites on either side of the product will work in theory. However the
attL sites are > 100 bases and hence very long PCR primers will need to be ordered. The oligos would probably have
greater propensity towards secondary structure, synthesis failure, etc. Hence such a strategy to generate an Expression
clone is not recommended.
Biologically an optimal LR reaction substrate is supercoiled attL with linear attR sites since helical density of the DNA is
important in lambda recombination. However, the LR reaction is a more effective reaction than the BP reaction it enables
the reaction to be done in a less than favorable condition and still achieves an acceptable amount of colonies. Hence both
the Entry clone and the destination vector can be present as supercoiled during the LR reaction. This will result in 2-5
fold less colonies than if the LR reaction was done with a supercoiled Entry clone and linear DEST vector.
The LR Clonase and reaction conditions
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LR clonase mix contains lambda recombination proteins Int (Integrase), Xis (Excisionase), and IHF (Integration Host
Factor).
The addition of Proteinase K is not necessary when doing an LR reaction. A typical LR reaction with Proteinase K
treatment yields about 35000 to 150000 colonies per 20 碌l reaction. Without the Proteinase K treatment there is an
approximate 10-fold reduction.
9
The success of the LR recombination reaction is very dependent on the molar ratio of the Entry clone and DEST vector.
If the ratio is not equimolar, the co-integrate may react with the DEST vector and Entry clone resulting in a higher
background.
Do not use too much DNA during transformation of E. coli after the LR reaction because the Entry clone (even if is
Kanamycin-resistant) may outgrow the DEST expression clone. The expression clone is usually in a pBR322-based ori,
which has a lower copy number for expression whereas the Entry clone has the high copy pUC ori.
Information on pENTR-gus
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This is the positive control for the LR recombination reaction.
The Gus protein has 603 amino acids; MW: 68.4 kDa. Gus refers to beta-Glucuronidase, a protein that can be detected
either with a fluorescent or blue substrate in the cells.
An LR reaction with pENTR-gus and a destination vector typically yields hundreds of colonies. The Gus gene has a Shine-
Dalgarno and Kozak sequence in frame with the attL1 site so this gene can be expressed either as native or fusion protein
in prokaryotic and eukaryotic cells.
Primers for sequencing Entry Clones:
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Clones derived from Sequence
SeqL-A (proximal to attL1) 5鈥橳CGCGTTAACGCTAGCATGGATCTC3鈥? (reads about
pDONR201, and pDONR207
60 bp of vector sequence)
SeqL-B (proximal to attL2) 5鈥橤TAACATCAGAGATTTTGAGACAC3鈥? (reads about
60 bp of vector sequence)
pDONR221 M13 Forward (-20): 5鈥橤TAAAACGACGGCCAG-3鈥?
M13 Reverse: 5鈥?-CAGGAAACAGCTATGAC-3鈥?
SeqL-C (proximal to attL1) 5鈥橤GATAACCGTATTACCGCTAG3鈥? (reads about 300
Entry Vectors pENTR1a, 2b,
bp of vector sequence)
3c, 4, and 11
Modified GW1 Forward primer: 5鈥橤TTGCAACAAATTGATAAGCAATGC3鈥? (reads
about 81 bp of vector sequence)
SeqL-B (proximal to attL2) 5鈥橤TAACATCAGAGATTTTGAGACAC3鈥? (reads about
70 bp of vector sequence)
SeqL-D (proximal to attL2) 5鈥橳CTTGTGCAATGTAACATCAG3鈥? (reads about 90 bp
of vector sequence)
SeqL-E (proximal to attL2) 5鈥橤TTGAATATGGCTCATAACAC3鈥? (reads about 170 bp
of vector sequence)
pCR8/GW/TOPO GW1 Forward: 5麓-GTTGCAACAAATTGATGAGCAATGC-3麓 (This primer can
anneal to attL1 sites except from pENTR1a, 2b, 3c, 4, and 11)
GW2 Reverse: 5鈥?- GTTGCAACAAATTGATGAGCAATTA-3鈥? (This primer can only
be used for pCR8/GW/TOPO since the TA in bold is unique to this vector)
pENTR/D-TOPO M13 Forward (-20): 5鈥橤TAAAACGACGGCCAG-3鈥?
M13 Reverse: 5鈥?-CAGGAAACAGCTATGAC-3鈥?
pENTR/U6 U6 Forward: 5鈥橤GACTATCATATGCTTACCG3鈥?
M13 Reverse: 5鈥機AGGAAACAGCTATGAC3鈥?
Sequencing the shRNA from pENTR/U6 or pENTR/H1/TO
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(back to Protocol and Application Notes)
10
The hairpin sequences are inverted repeats that form structures during sequencing. A drop in the sequencing signal has been
observed when entering the hairpin.
Suggestions:
1. Include more template and primer in the reaction (up to double the recommended amounts).
2. Longer hairpins or those with higher GC content tend to be more difficult to sequence. In situations like this it may help
to add up to 5-10% DMSO in the reaction.
3. Try different sequencing reaction enhancers; for example those which are used on high GC content
4. Use high quality template DNA; we use either Invitrogen's SNAP midiprep (Cat # K191001) or PureLink HQ miniprep
(Cat # K210001) kits to purify the DNA.
5. Work with the sequencing facility to get the right electronic file output. In our experience we often see a drop-off in
signal sequence after the start of the shRNA sequence (from either direction). There is often clear sequence there but
because the file output is calibrated to the stronger signals at the beginning of the read, the rest of the shRNA sequence is
compressed on the vertical axis. If the output is recalibrated to enhance the signals from the end of the shRNA, there may
be very good sequence information there (the signals from the beginning of the shRNA end up off the chart, so it may
require two different files to read the entire sequence). Hence the problem may not be bad sequence, but rather that there
are two distinct signal levels present, which cannot both be displayed in the same file.
6. If no other solution is possible, re-design the shRNAs to be more sequencing-friendly without a significant impact on
their potency. This strategy works well but should be a last resort, as it requires ordering new insert oligos. It can be
achieved in 2 ways:
Make 2-3 well-spaced base changes in the sense strand sequence. Changing an A to a G or a C to a T will break
up the inverted repeat in the DNA and will allow G:U basepairing in the shRNA. The shRNA will still form a
hairpin and the antisense strand will still perfectly match the target sequence. A good reference that describes
this strategy is Paddison et al. (2002). Genes Dev. 16(8): 948-58.
Change the loop to include a restriction enzyme site. Digest and purify the template at that site prior to
sequencing (e.g. on a PCR cleanup column such as the PureLink PCR purification kit; K310001) then use
forward and reverse primer reactions for each template. When the two parts of the shRNA-inverted repeat are
released from each other, the sequence data obtained is very good right up to the restriction cleavage site.
Primers for sequencing Expression Clones
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(back to Protocol and Application Notes)
Clones derived from Sequence
pDEST14 and pDEST17 ACG ATG CGT CCG GCG TAG AGG AT
pET-DEST42, pcDNA-DEST47, pcDNA- TAA TAC GAC TCA CTA TAG GG (Cat# N56002)
DEST53, pcDNA-DEST40, pEF-DEST51
pYES-DEST52 AAT ATA CCT CTA TAC TTT AAC GTC
pDEST8 and pDEST10 GTT CTA GTG GTT GGC TAC GTA TA (Note: This primer binds
before the polyhedrin promoter. For analyzing recombinant bacmid, this
primer along with a 3鈥? gene specific primer can be used)
pMT-DEST48 CAT CTC AGT GCA ACT AAA
pDEST 26 TGA ACC GTC AGA TCG CCT GGA GA
CGC AAA TGG GCG GTA GGC GTG
pT-REx-DEST30, pT-REx-DEST31
pDEST15, pDEST20, pDEST 27 GTG ATC ATG TAA CCC ATC CTG AC
pEF-DEST51 TCA AGC CTC AGA CAG TGG TTC
Sequencing the Destination vector after insertion of the Gateway Vector Conversion cassettes:
For cycle sequencing, it is best if the attR sites are on separate DNA fragments. DNA can digested to give two
fragments, each carrying one attR site. One of the restriction enzymes that can be used is AlwN I that cuts once in
the cassette upstream of the ccdB gene and usually cuts one or more times in the vector backbone. It does not matter
if more than two fragments are generated, as long as attR1 and attR2 are on separate DNA fragments. Following
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digestion, phenol extract, ethanol precipitate, and resuspend the DNA to ~200 ng/ul. Use 2.5 碌l in a 20 碌l Big Dye
sequencing reaction.
PRODUCT DOCUMENTATION
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Brochures Cell lines Citations
COA FAQ Licensing
Manuals MSDS Newsletters
Vector Data
COMPONENTS
Gateway Clonase Enzymes
(back to Table of Contents)
(back to Components)
Name Size Catalog Number
Gateway鈩? BP Clonase鈩? Enzyme Mix 20 rxns 11789013
100 rxns 11789021
BP clonase II enzyme mix (pre-mixed ready-to-use 20 rxns 11789020
solution of clonase and reaction buffer) 100 rxns 11789100
Gateway鈩? LR Clonase鈩? Enzyme Mix 20 rxns 11791019
100 rxns 11791043
Gateway鈩? LR Clonase鈩? Plus Enzyme Mix 20 rxns 12538013
LR Clonase II Enzyme Mix (pre-mixed ready-to-use 20 rxns 11791020
solution of clonase and reaction buffer) 100 rxns 11791100
Competent E. coli
(back to Table of Contents)
(back to Components)
Name Size Catalog Number
One Shot ccdB Survival T1 Phage-Resistant Cells 10 x 50 ul C751003
Donor vectors
(back to Table of Contents)
(back to Components)
Name Features Catalog Number
pDONR201 Kanamycin resistant 11798014
pDONR221 Kanamycin resistant 12536017
pDONR/Zeo Zeocin resistant 12535035
pDONR P2R-P3 Part of the MultiSite gateway 3-fragment 12537023
vector construction kit.
pDONR P4-P1R Part of the MultiSite gateway 3-fragment 12537023
vector construction kit.
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pDONR 222 Part of the CloneMiner cDNA library 18249029
construction kit. Kanamycin resistant.
Name Components Catalog Number
Gateway PCR Cloning BP Clonase, BP Clonase Rxn Buffer, pDONR221 Vector ,
system with 12535019
pEXP7-tet Positive Control, LE DH5伪飥燾ells, M13 primers.
pDONR221 vector
Gateway PCR Cloning BP Clonase, BP Clonase Rxn Buffer, pDONR/Zeo Vector ,
system with 12535027
pEXP7-tet Positive Control, LE DH5伪飥燾ells, M13 primers, Zeocin
pDONR/Zeo vector
Gateway PCR Cloning
BP clonase II, pDONR221, M13 sequencing primers, OneShot
System with clonase II 12535029
OmniMAX 2-T1 competent cells.
and pDONR221 vector
Gateway PCR Cloning
BP clonase II, pDONR/Zeo, M13 sequencing primers, OneShot
12535037
System with clonase II
OmniMAX 2-T1 competent cells.
and pDONR/Zeo vector
Entry vectors
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(back to Components)
Name Features Catalog Number
pENTR 1A N- and C-terminal fusions in E. coli or 11813011
eukaryotic cells
pENTR 2B N- and C-terminal fusions in E. coli or 11816014
eukaryotic cells
pENTR 3C N- and C-terminal fusions in E. coli or 11817012
eukaryotic cells
pENTR 4 N- or C-terminal fusions in E. coli. Native in 11818010
eukaryotic cells
pENTR 11 Native expression in E. coli or eukaryotic 11819018
cells.
pENTR/U6 RNAi studies using Lentiviral expression. K494500
pENTR/H1/TO Inducible RNAi studies using Lentiviral K492000
expression
pENTR221 Vector containing Ultimate ORF clone HORF01/MORF01
pENTR/GeneBLAzer Entry vector for the GeneBLAzer system 12578118
pCR8/GW/TOPO TA cloning vector, Spectinomycin selection K250020/
K252020
pENTR 5鈥? TOPO Entry vector for 5鈥? element in the MultiSite K59120
Gateway system
pENTR/D-TOPO Directional TOPO entry vector K240020
pENTR/SD/D-TOPO Directional TOPO entry vector with gene 10, K242020
and Shine-Dalgarno sequence
pENTR/TEV/D-TOPO Directional TOPO entry vector with 5鈥? TEV K252520
protease cleavage site
Destination vectors
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(back to Components)
Cell-free Expression
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Name Features Catalog Number
Expressway Plus In No vector (K990010), pEXP1-DEST K990010/
Vitro Protein Synthesis (K990020), or pEXP2-DEST (K990030) K990020/
System K990030
Expressway Plus No vector (K990060) or pEXP3-DEST K990060/
Expression System with (K990070) K990070
Lumio Technology
pEXP1-DEST N-terminal 6xHis, Xpress epitope, EK V96001
cleavage
pEXP2-DEST C-terminal V5-6xHis tag V96002
pEXP3-DEST N-terminal lumio, 6xHis tag, TEV, V96003
Prokaryotic Expression
Name Features Catalog Number
E. coli Expression Includes pDEST14, pDEST15, pDEST17, 11824026
System pDEST24, DH5a, BL21-AI, LR clonase
pET104.1-DEST. T7/lac promoter, N-term BioEase tag for in K10401
vivo biotinylation, EK cleavage
pET-DEST42 T7/lac promoter, C-term V5-6xHis. 12276010
pDEST14 T7 promoter, no tag 11801016
pDEST15 T7 promoter, N-terminal GST 11802014
pDEST17 T7 promoter, N-terminal 6xHis 11803012
pDEST24 T7 promoter, C-term GST 12216016
pET160-DEST T7/lac promoter N-terminal Lumio, 6xHis, 12583035
TEV site
pET161-DEST T7/lac promoter, C-term Lumio, 6xHis 12583043
pET160/GW/D-TOPO T7/lac promoter, N-term Lumio, 6xHis, TEV K16001
site
pET161/GW/D-TOPO T7/lac promoter, C-term Lumio, 6xHis K16101
pBAD-DEST49 N-term Thioredoxin, C-term V5-6xHis, 12283016
araBAD promoter
Yeast Expression
Name Features Catalog Number
pYES2-DEST52 GAL1 promoter, C-term V5-6xHis 12286019
pDEST22* Prey vector with the Gal4 AD in the 10835031
ProQuest two-hybrid system
pDEST32* Bait vector with the Gal4 BD in the ProQuest 10835031
two-hybrid system
Insect Expression
Name Features Catalog Number
Baculovirus Expression Baculovirus pDEST 8, 10, 20 set, LR 11827011
System with Clonase Enzyme Mix, pENTR-GUS Library
GATEWAY鈩? Efficiency DH5伪 cells
Technology
pDEST 8 Polyhedrin promoter, Native expression 11804010
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pDEST 10 Polyhedrin promoter, N-terminal 6X his 11806015
pDEST 20 Polyhedrin promoter, N-terminal GST 11807013
BaculoDirect N-Term Polyhedrin promoter, N-term 6xHis-V5, TEV 12562054/
linear DNA site 12562062
BaculoDirect C-Term Polyhedrin promoter, C-term V5-6xHis 12562013/
linear DNA 12562039
BaculoDirect Secreted Polyhedrin promoter, N-term honeybee 12562021/
linear DNA melittin (HBM) secretion signal, 6xHis-V5, 12562047
TEV site
pMT-DEST48 DES Metallothionein (MT) promoter, C-term 12282018
V5-6xHis
pMT/BioEase-DEST DES N-term BioEase tag for in vivo V414020
biotinylation, MT promoter, EK site,
pIB/V5-His-DEST OpIE2 promoter, C-term V5-6xHis, non-viral 12550018
stable insect expression
Mammalian Expression
Name Features Catalog Number
pcDNA3.1/nV5-DEST CMV promoter, N-terminal V5, Geneticin 12290010
selection
pcDNA3.2-DEST CMV promoter, C-terminal V5, Geneticin 12489019
selection
pcDNA6.2/V5-DEST CMV promoter, C-terminal V5, Blasticidin 12489027/
selection (Tag-on-demand technology) K42001
pcDNA6.2/GFP-DEST CMV promoter, C-terminal GFP, Blasticidin K41001
selection (Tag-on-demand technology)
pcDNA6/BioEase- CMV promoter, N-term BioEase tag, EK site K98001
DEST
pcDNA-DEST40 CMV promoter, C-term V5-6xHis, Geneticin 12274015
selection
pcDNA-DEST47 CMV promoter, C-term GFP, Geneticin 12281010
selection
pcDNA-DEST53 CMV promoter, N-term GFP, Geneticin 12288015
selection
pDEST26 CMV promoter, N-terminal 6xHis, Geneticin 11809019
selection
pDEST27 CMV promoter, N-term GST, Geneticin 11812013
selection
pT-REx-DEST30 CMV promoter, no tag, tetracycline inducible 12301016
system
pT-REx-DEST31 CMV promoter, N-term 6xhis, tetracycline 12302014
inducible system
pEF-DEST51 EF-1a promoter, C-term V5-6xHis, Geneticin 12285011
selection
pEF5/FRT/V5-dest Flp-In expression vector, EF-1a promoter, V- V602020
term V5, Hygromycin selection
Mammalian Expression With three destination vectors pcDNA3.2- 11826021
System with DEST鈩?, pDEST鈩?26, pDEST鈩?27 and
Gateway鈩? Library Efficiency DH5a, LR clonase,
pENTR-Gus, and Proteinase K.
pcDNA6.2/GW-V5/D- CMV promoter, C-term V5, Blasticidin K246020
TOPO selection
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pcDNA3.2/GW-V5/D- CMV promoter, C-term V5, Geneticin K244020
TOPO selection
pcDNA6.2/nLumio- CMV promoter, N-term Lumio, Blasticidin 12589032
DEST selection, Dual In-Cell Labeling kit
pcDNA6.2/cLumio CMV promoter, C-term Lumio, Blasticidin 12589016
DEST selection, Green In-Cell Labeling kit
pcDNA6.2/cLumio CMV promoter, C-term Lumio, Blasticidin 12589024
DEST selection, Red In-Cell Labeling kit.
pcDNA6.2/cGeneBLAz CMV promoter, C-term bla(M) tag, 12578043 in vivo
er-DEST Blasticidin selection, in vitro or in vivo 12578035 in vitro
detection kit
pcDNA6.2/nGeneBLAz CMV promoter; N-term bla(M) tag; 12578068 in vivo
er-DEST Blasticidin selection, in vitro or in vivo 12578050 in vitro
detection kit
pcDNA6.2/cGeneBLAz CMV promoter; C-term bla(M) tag; 12578084 in vivo
er-GW/D-TOPO Blasticidin selection, in vitro or in vivo 12578076 in vitro
detection kit
pcDNA6.2/nGeneBLAz CMV promoter; N-term bla(M) tag; 12578100 in vivo
er-GW/D-TOPO Blasticidin selection, in vitro or in vivo 12578092 in vitro
detection kit
Viral Expression
Name Features Catalog Number
pAd/CMV/V5-DEST Adenoviral expression, CMV promoter, C- V49320/
term V5, TK polyA K493000
pAd/PL-DEST Adenoviral expression, promoter-less, no V49420/
tags K4940-00
pAd/BLOCK-iT/V5- Adenoviral expression, promoter-less, use V49220
DEST with pENTR/U6 or pENTR/H1/TO entry
vectors.
pLenti 6/V5-DEST Lentiviral expression, CMV promoter, C- V49610/
term V5, Blasticidin selection K496000
pLenti4/V5-Dest Lentiviral expression, CMV promoter, C- V49810/
term V5, Zeocin selection K498000
pLenti 6/UbC/V5- Lentiviral expression, UbC promoter, C-term V49910/
DEST V5, Blasticidin selection K499010
pLenti4/TO/V5-DEST Lentiviral expression, CMV/TO tetracycline- K496700
inducible promoter, C-term V5, Zeocin
selection
pLenti 6/BLOCK-iT- Lentiviral expression for RNAi studies, use K494300/
DEST with pENTR/U6 or pENTR/H1/TO entry K494400
vectors, C-term V5, Blasticidin selection
pLenti 4/BLOCK-iT- Lentiviral expression for RNAi studies, use V48820
DEST with pENTR/U6 or pENTR/H1/TO entry
vectors, C-term V5, Zeocin selection
pLenti6/R4R2/V5- Promoter-less lentiviral expression, use with K591000/
DEST pENTR5鈥?-TOPO, no tags, Blasticidin K59110
selection.
Specialized vectors
Name Features Catalog Number
Gateway Vector Reading frame A, B and C.1 with one-shot 11828029
Conversion system ccdB survival T1 cells
pDEST R4-R3 Destination vector to be used in MultiSite 12537023
Gateway system
16
Gateway system
pCMVSPORT6 Not For mammalian library construction using 12209011
I/Sal I cut Superscript Plasmid
pBLOCK-iT 3 DEST Promoter-less vector for RNAi studies, no V48620
tags, use with pENTR/U6 or pENTR/H1/TO,
Geneticin selection
pBLOCK-iT 6 DEST Promoter-less vector for RNAi studies, no V48720
tags, use with pENTR/U6 or pENTR/H1/TO,
Blasticidin selection
pVAX200-DEST CMV promoter, designed for vaccine 12727010/
research, Kanamycin selection in E. coli 12727015/
12727023
pSCREEN-iT/lacZ- CMV promoter, N-terminal lacZ gene, V47020/
DEST reporter vector for screening RNAi K491500/
K491600
ASSOCIATED PRODUCTS
(back to Table of Contents)
Product Size Catalog Number
One Shot OmniMAX 2-T1R Chemically Competent E. coli 20 x 50 ul C854003
One Shot TOP10 Chemically Competent E. coli 20 x 50 ul C404003
Library Efficiency DH5alpha Chemically Competent E. coli 5 x 0.2 ml 11782018
One Shot ccdB Survival T1R Chemically Competent E. coli 10 x 50 ul C751003
S.N.A.P. MidiPrep Kit 20 reactions K191001
PureLink HQ Mini Plasmid Purification Kit 100 reactions K210001
PureLink PCR Purification Kit 50 reactions K310001
Ampicillin 20 ml (10 mg/ml) 11593019
Kanamycin Sulfate 100 ml (10 mg/ml) 15160054
Zeocin 1g R25001
5g R25005
AccuPrime Pfx DNA Polymerase 200 rxns 12344024
1000 rxns 12344032
AccuPrime Pfx SuperMix 200 rxns 12344040
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